The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.     

Immunomagnetic separation and analysis of non-malignant variants and parental malignant mouse lymphoma cells

We have devised conditions whereby non-tumorigenic, immunogenic cell variants of S49 mouse lymphoma were analyzed and separated from parental tumorigenic lymphoma cells. This was carried out using polyclonal antibodies (raised against the immunogenic variants) and immunomagnetic beads. The efficacy of the procedure depended on the amount of polyclonal antiserum, the immunobead to cell ratio, incubation time and the number of repetitions of the procedure. Experiments with mixed tumorigenic and non-tumorigenic cells have resulted in an enrichment of up to 200-fold of the non-tumorigenic, immunogenic cells in the population. These findings indicate the potential use of this procedure (in conjunction with other approaches) to isolate from a population of tumorigenic cells those variant cells that might be used to immunize against the parental tumor.     

Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline

cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murine cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.     

A comparison of the radiation sensitivities of non-tumorigenic and tumorigenic human hybrid cell lines

The radiation sensitivities of two related non-tumorigenic and two related tumorigenic human hybrid cell lines (HeLa x skin fibroblast) have been studied. The data show that the transformation from the non-tumorigenic to the tumorigenic state, which is accompanied by the loss of skin fibroblast chromosomes 11 and 14, is not associated with any major changes in radiation sensitivity. The data do indicate, however, a trend toward a steeper and longer initial slope to the cell survival curve for the tumorigenic cell lines, along with a subsequent reduced ability to accumulate sublethal radiation injury at low doses. Both nontumorigenic and tumorigenic cell lines have the capability of repairing sublethal injury.     

Transcriptional deregulation of the keratin 18 gene in human colon carcinoma cells results from an altered acetylation mechanism

We are investigating the mechanism responsible for the overexpression of the keratin 18 (K18) gene in tumorigenic clones from the SW613-S human colon carcinoma cell line, as compared with non-tumorigenic clones. We have previously shown that this mechanism affects the minimal K18 promoter (TATA box and initiation site). We report here that treatment of the cells with histone deacetylase inhibitors stimulates the activity of the promoter in non-tumorigenic cells but has no effect in tumorigenic cells, resulting in a comparable activity of the promoter in both cell types. The adenovirus E1A protein inhibits the activity of the K18 promoter specifically in tumorigenic cells. This inhibition can be reversed by an excess of CBP protein. The conserved region 1 (CR1) of E1A, which is involved in the interaction with the CBP/p300 co-activators, is necessary to the inhibitory capacity of E1A. A 79 amino acid long N-terminal fragment of E1A, encompassing the two domains of E1A necessary and sufficient for binding to CBP (N-terminus and CR1), has the same differential inhibitory capacity on the K18 promoter as wild-type E1A. Forced recruitment of GAL4-CBP fusion proteins to the K18 promoter results in a greater stimulation of its activity in non-tumorigenic than in tumorigenic cells. The histone acetyltransferase activity of CBP is essential for this differential stimulation and the presence of the CBP2 domain greatly augments the activation capacity of the fusion protein. Chromatin immunoprecipitation experiments carried out with anti-acetylated histone antibodies showed no difference in the level of histone acetylation in the region of the K18 promoter between the two cell types. The structure of chromatin in the promoter region is similar in tumorigenic and non-tumorigenic cells, as determined by mapping of DNase I hypersensitive sites and probing the accessibility of the DNA to restriction endonucleases. From all these results we conclude that alteration of an acetylation mechanism involving the CBP (or p300) protein and acting on a non-histone substrate is responsible for the higher activity of the K18 promoter in tumorigenic cells of the SW613-S cell line.     

c-AMP-induced c-fos expression in cells of melanocyte origin

The expression of the c-fos gene in murine cells of melanocyte origin in response to cAMP-elevating agents has been examined. Accumulation of c-fos mRNA at a high level as a consequence of these treatments precedes both proliferative and cytodifferentiative changes in non-tumorigenic or tumorigenic cell lines.     

Conversion of premalignant human cells to tumorigenic cells by methylmethane sulfonate and methylnitronitrosoguanidine

Nine human tumor cell lines derived from both epithelial and mesenchymal tumors exhibited either an anchorage-independent growth non-tumorigenic phenotype or an anchorage-independent tumorigenic phenotype. Transformed epithelial cell lines with the non-tumorigenic phenotype could be converted to a progressively growing tumor phenotype following treatment with either methylmethane sulfonate (MMS) or N-methyl-N-nitro-N-nitrosoguanidine (MNNG). In contrast, sarcoma derived cell lines with a non-tumorigenic phenotype could be converted to a progressively growing tumor phenotype only with MNNG. SV40 immortalized HET-1A non-tumorigenic phenotype cells could be converted to a progressively growing tumorigenic phenotype, infrequently, when treated with MNNG, but not MMS. Progressively growing tumors produced by either MMS or MNNG treated non-tumorigenic phenotypes exhibited metastatic potential in nude mice. Chemically treated HET-1A cells acquired the ability to produce tumor in mice but the tumor did not exhibit metastatic potential. In contrast, populations of tumorigenic cells were not rendered more biologically aggressive after treatment with either MMS or MNNG; i.e., the latency period for tumor development was not accelerated and the tumors did not exhibit metastatic potential. These results suggest that the biological effects of MMS and MNNG on non-tumorigenic, tumorigenic and immortalized cell lines are phenotype specific.Abbreviations AIG anchorage-independent growth - DMSO dimethyl sulfoxide - FBS fetal bovine serum - GM growth medium - MEM Eagle's minimum essential medium - MMS methylmethane sulfonate - MNNG N-Methyl-N-Nitro-N-Nitrosoguanidine - PDL population doubling - SCC squamous cell carcinoma     

Increased incidence of CAD gene amplification in tumorigenic rat lines as an indicator of genomic instability of neoplastic cells

It has been hypothesized that genomic instability is an important component of tumorigenesis. In an attempt to establish this relationship, we determined the frequencies with which two nontumorigenic and four tumorigenic rat liver epithelial cell lines underwent a particular type of genetic instability, gene amplification. By exposing cells to N-(phosphonoacetyl)-L-aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional CAD enzyme and selects for amplification of the CAD gene, we observed a striking parallel between the ability of these cell lines to become resistant to this drug and the ability of these same cells to form tumors after injection into day-old syngeneic rats. Cells of one highly tumorigenic line became resistant to PALA greater than 70 times more often than those of a non-tumorigenic line. Molecular analyses of eight independent PALA-resistant subclones confirmed that, in each case, this resistance was due to amplification of the CAD gene. Thus, our results demonstrate the relationship between tumorigenicity and at least one measure of genomic instability, CAD gene amplification. The method developed in this study provides a quantitative, rapid indicator of tumorigenicity and should prove useful in trying to elucidate the underlying basis of genomic instability in neoplastic cells.     

Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1).

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.     

Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA:m(6)A methyltransferase

MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m(6)A methyl-transferase (MTase), an enzyme that sequence-specifically methylates adenines in pre-mRNAs. The physiological importance yet limited understanding of MT-A70 and its apparent lack of similarity to other known RNA MTases combined to make this protein an attractive target for bioinformatic analysis. The sequence of MT-A70 was subjected to extensive in silico analysis to identify orthologous and paralogous polypeptides. This analysis revealed that the MT-A70 family comprises four subfamilies with varying degrees of interrelatedness. One subfamily is a small group of bacterial DNA:m(6)A MTases. The other three subfamilies are paralogous eukaryotic lineages, two of which have not been associated with MTase activity but include proteins having substantial regulatory effects. Multiple sequence alignments and structure prediction for members of all four subfamilies indicated a high probability that a consensus MTase fold domain is present. Significantly, this consensus fold shows the permuted topology characteristic of the b class of MTases, which to date has only been known to include DNA MTases.     

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.     

Novel Processed Form of Syndecan-1 Shed from SCC-9 Cells Plays a Role in Cell Migration

The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines.     

Epitope masking of rat esophageal carcinoma tumor-associated antigen by certain coexisting glycolipid and phospholipid molecules: a potential mechanism for tumor cell escape from the host immune responses

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.     

Coordinate N-ras mRNA up-regulation with mutational activation in tumorigenic guinea pig cells.

Tumorigenic guinea pig cell lines with mutationally activated N-ras alleles also exhibited up-regulated N-ras mRNA. Mutational activation and mRNA up-regulation were limited to tumorigenic cells; preneoplastic progenitors were unaffected. Therefore, up-regulation occurred at a late stage of carcinogenesis closely associated with acquisition of tumorigenicity. cDNA and S1 protection analysis demonstrated that polyadenylation site of the short N-ras message and the mRNA start sites were different from that reported for human. The promoter region contained no canonical TATA or CCAAT boxes, but exhibited GGGCGG and CCGCCC SPl binding motifs characteristic of growth control genes. Moreover, both mutant and wild-type alleles were up-regulated in a guinea pig line heterozygous for N-ras codon 61. Coordinate N-ras mutational activation and up-regulation in five independent tumorigenic lines with unique chromosome constitutions suggests that both events are required for expression of the neoplastic phenotype.